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首页> 美国卫生研究院文献>Biochemical Journal >Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.
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Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

机译：通过鼠嘌呤核苷磷酸化酶的活性位点修饰设计腺苷磷酸化酶。嘌呤核苷磷酸化酶Asn-243和Lys-244取代的酶动力学和分子动力学模拟。

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Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.
机译：我们的目标是改变嘌呤核苷磷酸化酶的底物特异性，以使其催化6-氨基嘌呤核苷的磷酸解。我们对Asn-243和Lys-244进行了修饰，以促进腺苷C6-氨基的接受。 Asn-243-Asp取代导致肌苷的K（m）增加了8倍，从58到484 microM，k（cat）/ K（m）减少了1000倍。 Asn-243-Asp构建体以45 microM的K（m）和肌苷的8倍的k（cat）/ K（m）催化腺苷的磷酸解。 Lys-244-Gln构建体仅显示k（cat）/ K（m）的少量减少，为野生型的83％，但对腺苷没有活性。与野生型相比，Asn-243-Asp; Lys-244-Gln构建体的K（m）随肌苷增加14倍，k（cat）/ K（m）减少7倍。这种双重取代以42 microM的K（m）和两倍于单一Asn-243-Asp取代的k（cat）/ K（m）催化腺苷的磷酸化。以腺嘌呤为底物的工程蛋白的分子动力学模拟显示，对于Asn-243-Asp和Asn-243-Asp; Lys-，嘌呤环的N7与Asp-243羧酸盐之间的氢键距离在2.93和2.88A。分别为244-Gln构建体。模拟还支持对于每个构建体，嘌呤C6-氨基和Asp-243之间的有利氢键距离分别为2.83和2.88A。 Asn-243-Thr取代不具有腺苷活性，并且模拟给出了Thr-243与嘌呤环的C6-氨基和N7之间不利的氢键距离。取代不在磷酸盐结合区域，并且具有野生型和Asn-243-Asp酶的磷酸盐的表观S（0.5）分别为1.35 +/- 0.01和1.84 +/- 0.06mM。两种蛋白均显示出与磷酸盐的正协同作用，希尔系数分别为7.9和3.8。

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